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Abstract

Mammalian cardiac myocytes (CM) proliferate readily during development but exit the cell cycle perinatally. The molecular mechanism for this is poorly understood could lead to new approaches for cardiac regeneration. We employed a novel bioinformatics approach to identify transcription factors that may control perinatal cell-cycle arrest and identified hypoxia inducible factor 1α (Hif1α) as a potential regulator of CM proliferation and differentiation. To test our hypothesis, e12.5d embryonic heart cells were cultured in atmospheric (17%) or hypoxic (3%) [O2] and then assessed for proliferation over 4 days. Total cell numbers were significantly increased (∼2 fold, P<0.005) in cells cultured in 3% vs. 17% [O2] after 4 days. Hypoxia increased numbers of cTn+ cells labeled with p-Histone-H3 indicating an increase in CM proliferation. Carboxyfluorescein succinimidyl ester staining indicated that the majority (70%) of cells cultured in 3% [O2] divided 2–3 times while the majority (73%) of cells in 17% [O2] divided 0–1 times after 3 days of culture. As expected, hypoxia stabilized the Hif1α protein and induced the expression of Vegf. Messenger RNA for the G1-S phase cyclin dependent kinase Ccnd2 was increased (>2 fold) in hypoxic vs. atmospheric cultured cells indicating that hypoxia my increase proliferation by regulating the expression of cell cycle machinery. Furthermore, early mesodermal transcription factors Brachyury and Mesp1 and the CM progenitor marker Nkx2.5 were induced (>5 fold) in hypoxic vs. atmospheric [O2] cultured cells suggesting that hypoxia positively regulates early mesodermal and CM progenitor cell differentiation. To validate this hypothesis, we differentiated mouse embryonic stem cells in atmospheric and hypoxic [O2] and observed similar increases in the expression of Brachyury, Mesp1 and Nkx2.5 in hypoxic differentiated cells. Transient culture (2 d) of embryoid bodies (EBs) in hypoxia increased the frequency of beating EBs and the % of cTn+ cells compared to cells differentiated in atmospheric O2. Taken together these data suggest that the hypoxic environment in which CMs develop may serve as a key signal that regulates mesodermal progenitor marker expression and CM progenitor cell differentiation and proliferation.